Stable, covalently-bonded supports for chemical separation apparatus made through a hydride intermediate

ABSTRACT

The present invention produces very stable, covalently bonded separation substrates for separations application such as liquid and gas chromatography as well as capillary zone electrophoresis. An intermediate substrate is prepared which has hydride species on the substrate surface. These hydrides preferably are further derivatized by the catalytic addition of organic compounds bearing a terminal vinyl group. The final surface modification contains closely packed, direct carbon linkages that are stable.

This is a continuation-in-part of application Ser. No. 07/407,816, filedSep. 15, 1989 now U.S. Pat. No. 5,017,540.

FIELD OF THE INVENTION

This invention relates to a surface-modified material used in a widevariety of separation applications such as chromatography andelectrophoresis.

More particularly, the invention pertains to a chemically modifiedmineral oxide such as silica, quartz or the like, which exhibitsimproved hydrolytic stability, larger organic coverage and superiorseparative capabilities when formed into various forms or shapes, suchas porous beads or capillary tubes.

BACKGROUND OF THE INVENTION

Chemically modified silicas have been, and continue to be, widely usedas supports in a great variety of chromatographic separations. With theaim of controlling its selectivity while reducing unwanted interactionswith one or more compounds, numerous synthetic procedures have beendeveloped to attach organic moieties (R) on the silica surface. Earlywork on the chemical modification of silica (Halasz and Sebastian,Angew. Chem. (Int. Ed.) 8:453 (1969); Deuel et al., Helv. Chim. Acta119:1160 (1959)) described the use of an esterification reaction betweensurface silanol groups (SiOH) and an alcohol to give a structure of thefollowing type: ##STR1## Although such materials were useful for manyseparations, their limited hydrolytic stability seriously precluded theextensive usage of these bonded phases, particularly in liquidchromatography which requires the use of aqueous eluents.

Currently, commercially available bonded phases are prepared by reactingselected organosilanes with the silica surface. Halogen- oralkoxy-substituted alkyldimethylsilanes are the most commonly usedsilanizing reagents. The resulting bonded support bears monolayersurface structures of the following type: ##STR2## By changing thestructure of the R group, it is possible to produce bonded silicas witha great variety of organic groups, ranging from non-polar materials, forinstance, octyl- and octadecyl-silicas commonly used as bonded supportsin reversed-phase liquid chromatography, to ionic materials such asbenzenesulphonic acid derivatives which are widely used in ion-exchangeliquid chromatography. The preparation of these and similar materialsare described in a number of publications (e.g., Roumeliotis and Unger,J. Chromatogr. 149:211 (1978) or Asmus et al. J. Chromatogr. 123:109(1976)) and patents (Sebastian et al. U.S. Pat. No. 3,956,179; Hancok etal. U.S. Pat. No. 4,257,916; or Ramsden et al. U.S. Pat. No. 4661,248).

In a related approach, polymeric (multilayer) bonded stationary phasesare prepared from bi- or tri-substituted organosilanes with the generalformula X_(n) SiR_(4-n), where X=alkoxy, halide or any easily hydrolyzedgroup, and n=2,3. The resulting polymeric bonded support bears repeatingsurface structures of the type ##STR3## where Y=--R (n=2) or --O-- (n=3)and the oxygen atom (--O--) is bonded either to a hydrogen (that is, aspart of a free silanol, Si--O--H) or to another silicon atom (that is,as part of a siloxane linkage, Si--O--Si). A number of patents andpublications describe the preparation of these materials (Kirkland andYates, U.S. Pat. Nos. 3,722,181 (1973), and 3,795,313 (1974); Novotny etal., J. Chromatog. 83:25 (1973); Sander and Wise, Anal. Chem. 56:504(1984)). Although in many instances these bonded supports providesatisfactory separations, the lack of control of the polymerizationprocess seems to be a major contributor to such problems asirreproducible layer thickness and incomplete silanol condensation. Thislimitation has confined polymeric bonded stationary phases toapplications where the presence of a multilayer is necessary and/or itsthickness is relatively unimportant. As a consequence, the vast majorityof liquid chromatographic separations are carried out with monolayerbonded phases.

The recent development of electrophoretic separations in a capillaryformat has promoted the extent of the silanization technology normallyused in chromatography to the deactivation of the inner wall of thefused silica capillary. Thus, Jorgenson et al. (Science 222:266 (1983))have noted that separation of model proteins, such as cytochrome,lysozyme and ribonuclease A, in untreated fused silica capillaries witha phosphate buffer at pH 7 was accompanied by severe tailing, andsuggested that this might be caused by strong interactions between theproteins and the capillary wall. Derivatization of the capillary wallhas been proven effective to prevent or control protein adsorption(McCormick, Anal. Chem. 60:2322 (1988); Bruin et al., J. Chromatog.471:429 (1989)). In addition, by chemically modifying the inner surfaceof the capillary, operational variables such as the electrosomotic floware more amenable to control. In another application (Hjerten, U.S. Pat.No. 4,680,201 (1987); Cohen and Karger, U.S. Pat. Nos. 4,865,706 and4,865,707 (1989)), a method is described for preparing fused-silicacapillary tubes for electrophoretic separations by use of a bifunctionalcompound in which one group (usually a terminal --SiX₃ group whereX=ethoxy, methoxy or chloride) reacts with the capillary wall and theother (usually an olefin group) does so with a monomer taking part in apolymerization process. This process resulted in a wall-bonded,polymer-filled capillary useful for polyacrylamide gel electrophoresis.

The extensive usage of these bonded materials in chromatography andcapillary electrophoresis does not necessarily imply that they meet allrequirements with respect to separation performance and stability. Onthe contrary, monomeric bonded phases, for instance, are subject toserious effects arising primarily from a relatively limited organiccoverage due to the "bulky" methyl groups of the anchored moiety, andfrom a still unsatisfactory hydrolytic stability of the Si--O--Si--Clinkage, particularly under moderately acidic or slightly alkalineelution conditions. Similarly, polymeric bonded phases although havingsomewhat better organic coverages, contain a considerable population offree silanols and also exhibit a limited hydrolytic stability.Incomplete surface coverage and poor hydrolytic stability both result inthe exposure of a substantial number of surface silanols, groups whichare known to be primarily responsible for the residual adsorptionphenomena that plague silicon-based separation materials. These socalled "silanophilic" interactions are usually undesirable inchromatography as well as in capillary electrophoresis because theyoften result in "tailing" peaks, catalyze solute decomposition, lead tounreliable quantitation, etc. One of the most striking cases ofsilanophilic interactions occurs perhaps in the separation of certaincompounds containing amino or other similar groups, particularlybiomolecules. For instance, many proteins may interact very stronglywith unreacted silanols leading to excessive band tailing, incompleterecovery of one or more solutes, or even recovery of the same componentfrom different bands.

In an effort to overcome such problems, other organosilane reagents havebeen developed. Two related approaches have been proposed in whicheither the methyl groups of the organosilane reagent are replaced bybulkier groups (Glajch and Kirkland, U.S. Pat. No. 4,705,725, (1987)) ora "bidentate" silanizing reagent is used (Glajch and Kirkland, U.S. Pat.No. 4,746,572, (1988)). In both cases the new groups are aimed to shieldthe unreacted silanols as well as the hydrolytically labile linkage thatbonds the silane to the support. Although this steric protection hasresulted in somewhat improved bonded phases, the synthetic proceduresstill involve the formation of unstable Si--O--Si--C linkage, andtherefore, the necessity still exists for a truly effective silanechemistry.

In another completely different approach, bonded silicas bearing directSi--C linkages have been developed. They involve the sequential reactionof the silica substrate with a chlorinating reagent (e.g., thionylchloride) and a proper alkylating reagent (e.g., a Grignard ororganolithium compound): ##STR4## where --M=--Li or --MgBr. Inprinciple, this method should provide not only a closer attachment and adenser coverage of organic functionalities but also a morehydrolytically stable bonded phase than that obtained by thecorresponding Si--O--Si--C linkage. However, the acceptance for theapplication of a chlorination/Grignard or chlorination/organo-lithiumreaction sequence as a routine method to modify silica substrates hasbeen hindered by several factors. One factor is that the one-steporganosilanization procedure (such as described in U.S. Pat. No.3,956,179 to Sebastian et al.) is relatively easy to carry out ascompared to the two-step halogenation/alkylation sequence. Difficultiesassociated with the removal of residual salts which may be occluded inthe porous silica matrix during the alkylation process is also animportant factor which has contributed to the limited usage of thissynthetic approach. Finally, but not less importantly, the preparationof the alkylation reagent exhibits strong interferences with manyreactive functionalities, particularly those containing carbonyl,nitrile, carboxyl, amide, alcohol, etc. That is, the great reactivitywhich makes a Grignard reagent so useful in many synthetic approachesseriously limits its applicability. The organic group, R, in theGrignard reagent, RMgBr, must remain intact during the preparation ofthe reagent. It is a well known fact that Grignard reagents react withacidic components to form the corresponding hydrocarbon group R--H. Morestrictly, "any compound containing hydrogen attached to anelectronegative element such as oxygen, nitrogen, and even triply-bondedcarbon are acidic enough to decompose a Grignard reagent" (Morrison andBoyd, Organic Chemistry, 3rd Edition, 1974). Additionally, a Grignardreagent reacts readily with molecular oxygen, carbon dioxide, and with"nearly every organic compound containing a carbon-oxygen orcarbon-nitrogen multiple bond" (supra). The nitro group (--NO₂) alsoappears to react oxidatively with a Grignard reagent. It seems cleartherefore that only a very limited number of organic functionalities maybe present in the halide compound from which a Grignard reagent can beprepared. Being even more reactive than the corresponding Grignardreagent, an organolithium reagent should exhibit the same limitationsdescribed above to a similar or even greater extent. This, of course,greatly limits the versatility of this approach.

It is therefore desirable to address the shortcomings of existing bondedpackings by developing an alternate silane chemistry which combines thesuperior coverage and hydrolytic stability of direct Si--C linkages withthe preparation simplicity of silanization.

SUMMARY OF THE INVENTION

In one aspect of the present invention, a solid intermediate is providedthat comprises an inorganic oxide-based, relatively rigid surface. Thesurface (after final derivatization) is exposable to fluids withcomponents therein being separated, such as during chromatographic orelectropheric separations. The intermediate surface before finalderivatization has hydride groups thereon.

After final derivatization, supports of the invention have an inorganicoxide substrate to which is covalently attached an organicfunctionality, through hydrolytically stable surface-to-carbon linkages.A preferred support of this invention comprises a silica substratewhich, upon derivatization by methods described in this invention,contains surface structures of the following type: ##STR5## where R isan alkane, substituted alkane, alkene or substituted alkene.

The present invention represents a totally different approach to theprior problems in producing very stable, covalently bonded separationsubstrates for all types of liquid and gas chromatography as well ascapillary electrophoresis.

Supports of the invention are prepared by the catalytic addition ofsilicon hydrides to organic compounds bearing a terminal vinyl oracetenyl group via a solid intermediate, which provides the siliconhydride species on the substrate surface. The final product containsclosely packed direct silicon-carbon linkages thus providing asignificantly improved surface-modified separation support with regardto stability and silanophilic interactions. Additionally, because of theintrinsic freedom from interferences of the catalytic SiH addition(hydrosilation), the method of preparation is an extremely versatile onein that it allows bonding of virtually any organic functionality to asupport material, in a clean, high-yield procedure. By properly choosingthe chemical composition of the R-group, chemically bonded separationmaterials may be prepared which exhibit a wide range in selectivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates partial IR spectra of hydride intermediates preparedaccording to the invention via a chlorination/reduction sequence, asdescribed in Example 1 (curve A); or by coupling with the acidhydrolysis product of triethoxysilane, as described in Example 2 (curveB);

FIG. 2 illustrates partial IR spectra of octyl- (curve A)andoctadecyl-(curve B) bonded silicas prepared according to the invention,as described by Example 3;

FIG. 3 illustrates plots of surface coverage versus hydrolysis time foroctyl bonded Vydac 101TPB™ silicas, one of which (hydrosilation product)was prepared according to this invention and the other (silanizationproduct) from a commercial (prior art) procedure. The long-termhydrolysis was in 15 mM trifluoroacetic acid solution containing 10% v/vdioxane, as described in Example 4;

FIG. 4 is analogous to FIG. 3, but is shown on a relative basis; and

FIGS. 5 and 6 illustrate the same test as FIGS. 3 and 4, but this timethe test solution was 15 mM phosphate at pH 2.0.

DETAILED DESCRIPTION OF THE INVENTION

This invention differs from most of the materials currently available byincluding a direct substrate-to-carbon bond instead of asubstrate-O--Si--C type of linkage. It is a primary purpose of thisinvention to provide a surface-modified separation material whichexhibits extended lifetime (hydrolytically stable), displays improvedadsorption properties (more extensive and versatile organic coverage),and is substantially free of contaminants (e.g., residual salts and thelike).

This unique material of the invention is produced by the catalyticaddition of surface hydride species to an organic reagent containing amultiple carbon-to-carbon bond, after converting the original surfacehydroxyl to hydride groups. The method of making this surface-modifiedmaterial is a very versatile one in that it allows the attachment to asubstrate of organic functionalities which could not be possible byregular organometallic procedures.

Supports of the invention are produced from a structurally rigidinorganic oxide which provides a hydrated surface vastly populated byhydroxyl groups. Suitable inorganic substrates as precursors include,but are not limited to, oxides of metalloids and metals such as silicon,aluminum, tin, thorium, magnesium, titanium, zirconium, etc., andcombinations thereof. In a preferred embodiment, the substrate precursormaterial is silicon oxide in the form of silica, quartz or the likematerials which are commonly used in gas and liquid chromatographic aswell as in capillary electrophoretic separations. In two particularlypreferred embodiments, the substrate precursors to be modified areporous, particulate silica (such as beads), as well as non-porous, fusedsilica or quartz capillary tubes.

The products of the present invention are prepared by a modificationscheme which comprises two major steps: (1) Attachment of hydridespecies on the substrate precursor so as to give a fairly stableintermediate; and (2) Reacting said hydrided surface with organiccompounds bearing a terminal unsaturated hydrocarbon group, in thepresence of a catalyst, whereby direct linkage of said inorganicsubstrate to carbon is provided.

The hydroxyl groups of the oxide substrate precursor provide activesites which can be chemically transformed into intermediate surfacehydride groups. This is accomplished either by direct conversion of thehydroxyl groups to hydride groups via a halogenation/reduction sequence,or more preferably, by condensation of the surface hydroxyl groups witha hydridosilane coupling reagent. In the latter instance, the hydrideintermediate is thus obtained as a surface deposition of thetrihydroxysilane hydrolysis product from a hydrolyzable trisubstitutedsilane. However, in either case, a chemically and thermally stablehydride intermediate is obtained in which most of the original hydroxylgroups are replaced by silicon hydride species.

In one procedure the substrate precursor material, e.g., silica, isfirst reacted with a suitable excess of a halogenating reagent,preferably thionyl chloride, in the presence of an anhydrous solventsuch as toluene: ##STR6## Reduction of the halogenated material is theneffected by reaction with a suitable excess of a solution of a metalhydride such as lithium aluminum hydride or its derivatives: ##STR7##The reduced material is finally subjected to a "clean-up" step with adilute aqueous acid, so as to remove chemisorbed salts (e.g., aluminumchloride) which originate from the reducing reagent.

A more preferred procedure to prepare the hydride intermediate involvesthe reaction of the inorganic oxide substrate, e.g., silica, with asuitable excess of a hydridosilane ethereal solution containing dilutemineral acid (typically 0.1 M HCl): ##STR8## where X is a hydrolyzablegroup, preferably an alkoxy or halide group such as ethoxy or chloride.

The hydrided surface is then reacted with an organic compound containinga carbon-to-carbon multiple bond, preferentially a terminal vinyl group,in the presence of an appropriate catalyst so as to give a directlinkage of the surface to carbon: ##STR9## where n≧0 and R'=alkane,substituted alkane, alkene or substituted alkene That is, R' ranges fromsimple hydrocarbon groups such as n-alkyls to heteroatom compounds suchas carbonyls, nitriles, amides, epoxy, etc., depending on theapplication for which the final addition product --the bondedsubstrate-- is intended. Alternatively, an acetenyl-terminated compoundwith the general formula HC═C--(CH₂)_(n) --R' can be used.

As previously known in other contexts, the addition of silicon hydridesto unsaturated hydrocarbons, commonly referred to as hydrosilation orhydrosilylation, has been recognized as one of the most importantlaboratory methods to form Si--C bonds. The reaction's minimalinterference with other reactive functionalities (e.g., CO₂ R, CN, NH₂,etc.) has permitted the attachment of silicon to organic molecules whichotherwise cannot be introduced by regular organometallic procedures.Details of the reaction in homogeneous phase can be found elsewhere(e.g., Speier, Adv. Organomet. Chem. 17:407 (1979); Seyferth, editor, J.Organomet. Chem. Library 5:1 (1977)).

Hydrosilation is generally carried out in the presence of a metalcatalyst. A variety of inorganic and organic complexes of transitionmetals such as platinum, rhodium, palladium, ruthenium, iridium andnickel have functioned as very effective catalysts for the additionreaction. The catalyst often consists of a solution of a halide-,olefin-, carbonyl- or phosphine complex of the transition metal.Chloroplatinic acid in an isopropanol solution (also known as "Speiers"catalyst) is the most commonly used form. Only as little as 10⁻⁵ mole ofplatinum per mole of silicon hydride is normally sufficient for aneffective hydrosilation. Commonly, an "induction period" is requiredwhen the Speiers catalyst is used. The addition then becomes rapid andcan be done at room temperature or under reflux to ensure a high yield.

For simple liquid olefins no additional solvent is normally required.For highly reactive olefins (such as those with a strong tendency topolymerize, e.g., allylmethacrylate, allyl glycidoxyl ether, etc.) aninert solvent such as toluene, benzene, saturated hydrocarbons,chloroform, etc. is suitable. In general, the reaction is convenientlycarried out under dry conditions, at temperatures often below theboiling point of the liquid. Typically, an excess of the olefin withrespect to the available surface hydride groups is used. The magnitudeof such an excess depends on the nature of the substituents in theolefin. Highly reactive reagents (epoxy-containing olefins, forinstance) require 10 to 50% molar excess while simple (unsubstituted)olefins may need a 10-fold molar excess or more to produce a highsurface coverage.

As with any surface modification procedure, the sites of the bondingreaction will eventually become sterically hindered at some point and,consequently, not all of the Si--H sites will be converted to Si--C. Inorder to remove as many of the remaining hydrides as possible, a"hydride end-capping" procedure usually follows the primary bondingreaction. Ethylene gas is conveniently used for this purpose since itoffers the smallest possible steric hindrance in olefinic addition. Oncethe main bonding reaction is considered complete, the ethylene gas isintroduced into the reactor and maintained at high pressure over thesolution containing the bonded support. This mixture is stirred andheated again for a period of several hours. The need for hydrideend-capping is particularly critical when aqueous alkaline solutions areused. Under these conditions, hydride groups are rapidly hydrolyzedgenerating hydrogen gas, with obviously deleterious effects if thereaction occurs during the course of a separation. Under acidicconditions, on the other hand, silicon hydride groups are virtuallyindefinitely stable and, therefore, hydride end-capping may not benecessary. Compared to conventional silanol end-capping of the prior artin which a bulky trimethylsilyl group, (CH₃)₃ Si--, is attached, hydrideend-capping in the present invention results in a "skinny" (linear)ethyl surface ligand, CH₃ CH₂ --. A more efficient secondary surfacecoverage can therefore be expected.

In general, the hydrosilation reaction has a great deal of versatility.This is due to the fact that relatively few reactive functionalitiesinterfere with the olefinic addition. For example, hydrosilationscatalyzed by chloroplatinic acid have been used to attach to siliconorganic groups containing a wide variety of functionalities including:halogens, nitrite, cyanide, amines, alkylsulfites, alkylsulfonamides,borate esters, and phosphohalides. The ester group (--CO₂ R) does notnormally interfere with the hydrosilation reaction. However, addition tothe carbonyl group of aldehydes and ketones usually, although notalways, takes place. This seems to be particularly true forα,β-unsaturated carbonyls. A similar behavior is exhibited byunsaturated nitriles as well as epoxydes of 1,3-dienes. By using olefinswhose C═C bond is separated from the heteroatom unsaturation by at leasta methylene (--CH₂ --) group, normal 1,2-addition is readily achieved.Carboxylic acids, phenols and alcohols react at the --OH group, althoughnormal addition can occur with olefinic tertiary and sometimes secondaryalcohols in which alcoholysis of the Si--H group is sterically hindered.Acidic functionalities can be bonded to silicon, however, by"protecting" the --OH group with a (CH₃)Si-- group, which can be readilyhydrolyzed off afterwards.

Among the major advantages of the practice of the present invention isthat the relatively limited surface coverage due to the "bulky" methylgroups in prior art organosilanes can be avoided and, consequently, amore densely populated surface can be obtained. Additionally, thehydrolytic advantage of direct Si--C linkages may be achieved withoutthe disadvantages that occur when such a linkage is obtained by theknown sequential reaction with a chlorinating reagent and an alkylatingreagent such as Grignard or organolithium. Moreover, the intrinsicfreedom from interference makes hydrosilation a particularly convenientapproach to attach virtually any organic functionality to a hydridesupport, resulting in a remarkably versatile separation material. Thus,the present invention not only combines the superior coverage andhydrolytic stability of direct Si--C linkages with a simplicityapproaching that of currently available silanization procedures, butalso provides a versatile separation support suitable for virtually anyapplication.

The primary applications for the invention are in the areas of bondedphases for high performance liquid chromatography (HPLC) as well asinner surface-modified capillaries for high performance capillaryelectrophoresis (HPCE). In both applications the products of theinvention will be especially useful for the separation of biologicallyimportant solutes such as proteins and nucleic acids as well as theirfragments. Very often, HPLC separations of proteins have to be carriedout in mobile phases containing aggressive electrolytes at low pH, suchas trifluoroacetic acid. Under these conditions bonded supports from theprevious art perform poorly with reference to organic phase degradation.Similarly, HPCE separations of proteins in surface-modified capillariesproduced by the previous art exhibit "bleeding" of the bonded coating.In any case, this results in either irreproducible results on ananalytical scale or in a significant solute contamination on apreparative scale. The products of this invention overcome thedegradation disadvantage of prior HPLC and HPCE materials while stillproviding similar or better separation performance.

In its most preferred form, the organic reagent used in the presentinvention assumes the general formula CH₂ ═CH--(CH₂)_(n) --G, where theterminal vinyl group provides attachment to the support surface, thegroup --G provides the desired functionality, and the value of ncontrols the length of the chain separating the two groups. The bondedsupports of this invention can contain a wide variety of functionalgroups to fit virtually any application. A few illustrative examples inHPLC include non-polar, purely hydrocarbonaceous --G ligands for"reversed" phases, polar groups such as nitrile (--C.tbd.N) for "normal"phases, and ionogens such as quaternary ammonium salts and sulfonicacids for anion- and cation-exchange phases respectively.

A particularly important application of the present invention is inaffinity chromatography. In this case, the --G functionality assumes aspecially reactive form such as epoxy or certain succinimido esters,represented by the following substituted-olefin structures: ##STR10##Once bonded to the hydrided substrate, these groups are readily bound toligands with specific biochemical activity. Of special novelty is thereactive ester because the current art requires a cumbersome, multi-steppreparation procedure.

For size-exclusion chromatography, the --G functionality takes the formof hydrophilic groups such as polyols, which can be readily prepared viaacid hydrolysis of previously bound epoxy groups. Alternatively theepoxy ring can be opened with a properly sized polyglycol, in which casea polymeric coating will result.

A similar polyol-modified surface can be used in open-tube capillaryelectrophoresis. In this case the hydrophilic fused-silica capillary isparticularly useful for the electrophoretic separation of biopolymerssuch as proteins and their fragments. In another important HPCEapplication, an olefinic modifier such as allylmethacrylate (in which--G=--O--CO--C(CH₃)═CH₂) can be bonded to the capillary wall. Thisbonded functionality is then copolymerized with a gel mixture to producea gel-filled, wall-bonded capillary which is useful for gel HPCE.Extremely high resolving power is achieved with these capillaries whenapplied to polyacrylamide gels. This technique is particularlywell-suited for the separation of biologically important macromoleculesincluding nucleic acids, proteins and their fragments, under bothdenaturing as well as non-denaturing conditions.

EXPERIMENTAL Materials and Methods

Toluene, diethyl ether and dioxane (EM Industries, Inc.) were dried byallowing them to stand with calcium hydride (Sigma Chemical Co.) forseveral days, refluxing and then distilling from the hydride immediatelybefore use. A 0.2 M lithium tetrahydridoaluminate (Sigma Chemical Co.)ether solution was prepared and used as the reducing reagent. Thionylchloride ("Gold Label" Aldrich Chemical Co ), 1-octene and 1-octadecene(Sigma Chemical Co.) were used as received. Infrared quality potassiumbromide (Harshaw/Filtrol Partnership) powder was used for the FT-IRspectra. Two particulate silica substrates were used: Partisil-40™(Whatman Inc., Clifton, N.J.) with a 40 μm mean particle size, 85Å meanpore diameter and 315 m² /g surface area; and Vydac 101TPB™ (TheSeparations Group, Hesperia, Calif.) with 5.6 μm mean particle size,334Å mean pore diameter and 89 m² /g surface area.

All silica derivatization reactions were carried out under a drynitrogen atmosphere in glassware that had been previously dried at 120°C. overnight. Transfer of liquids was accomplished either with a glasssyringe (<20 mL) or by means of a stainless steel cannula and nitrogenpressure, via silicone rubber septa. Prior to reaction, the silicasubstrate was dried under vacuum at 110° C. overnight and then cooled ina vacuum desiccator.

Infrared spectra were taken in the 4,000-450 cm⁻¹ region with a PerkinElmer Model 1800 FT-IR spectrometer equipped with a Spectra-Tech diffusereflectance accessory. Silica samples were mixed 1:1 by weight with KBrand 100 sample scans were ratioed against pure KBr as a reference.Spectra shown were normalized to 100% transmittance.

EXAMPLE 1 Preparation of Hydride Silica by a Chlorination/ReductionSequence

5.00 g of dried Partisil-40™ silica were suspended in 60 mL of freshlydistilled, dry toluene, and 10 mL of thionyl chloride (to obtain a10-fold ratio excess with respect to silanol content) were added. Themixture was magnetically agitated and the chlorination was allowed toproceed under reflux for at least 18 hours after which the excess SOCl₂was distilled off. Removal of any remaining SOCl₂ was achieved bywashing the dark-purple product at least 8 times with 80-mL portions ofdry toluene while magnetically stirring for 15 min. After each washing,and once the solid had settled, the solvent was carefully aspirated offto waste by means of a slight vacuum applied to a glass pipette.Finally, the chlorinated silica was washed with one 30-mL portion of drydiethyl ether, and then left in a final fresh ether aliquot.

70 mL of 0.2 M LiAlH₄ ether solution (about a 4-fold molar ratio,hydride/original silanol) were added slowly to the chlorinatedsilica/ether suspension while stirring. An immediate reaction wasevidenced by a color change from dark purple to white. The reaction wasallowed to proceed for two hours under a gentle reflux. A dry-icecondenser was found to be appropriate to safely condense relativelyvolatile intermediate reaction byproducts. The excess of LiAlH₄ was thenaspirated off and destroyed by adding ethyl acetate (about 10 mL)followed by isopropanol dropwise with stirring until hydrogen evolutionceased. The product was next washed with eight 30-mL portions of dryether to remove any remaining aluminum hydride and/or chloride speciesin solution. The "hydrided" silica was then dried overnight in a vacuumdessicator at room temperature. The dry solid was washed 3 times with 50mL portions of a 0.5 M HCl aqueous solution, followed by furtherwashings with tetrahydrofuran (THF)/water 1:1 v/v and ether. The productwas finally dried at 110° C. under vacuum overnight. The partial IRspectrum of the hydride intermediate so prepared is illustrated by curveA of FIG. 1.

EXAMPLE 2 Preparation of Hydride Silica by Silane Condensation(Coupling)

Five grams of Partisil-40™ silica were suspended in 50 mL of dioxanecontaining 5 mL of 3 M HCl aqueous solution. The suspension wasmagnetically stirred, heated at about 75° C. and then 110 mL of a 0.2 Mtriethoxysilane solution in dioxane were added dropwise (obtaining about30% molar excess of silane with respect to silanol). The reaction wasallowed to proceed under reflux for about 60 minutes after which thesuspension was centrifugated and the solid washed consecutively with50-mL portions of 1:1 v/v THF/water, THF and diethyl ether. Aftersolvent removal, the solid was dried at 110° C. under vacuum overnight.

Except for a favorably higher Si--H surface concentration, the productobtained exhibited essentially identical spectroscopic, chemical andthermooxidative characteristics to that prepared viachlorination/reduction sequence as described in Example 1. Thesimplicity and efficiency of this procedure make the silane coupling apreferred method. The partial IR spectrum of the hydride intermediate soprepared is illustrated by curve B of FIG. 1.

EXAMPLE 3 Preparation of Octyl- and Octadecyl-Bonded Silicas

60 mL of 1-octene (density 0.715 g/cc, 97% purity) containing 75 μL of0.1 M chloroplatinic acid solution in 2-propanol were heated to about70° C. while agitating magnetically for about 30 min or until a clearsolution was obtained. Five grams of hydride intermediate substrateprepared as described by Example 1 were then added to theolefin/catalyst solution, and the reaction allowed to proceed for about24 hours at 100±2° C. The mixture was then centrifugated and the solidwashed with three 40-mL portions of toluene followed by similar washingswith dichloromethane and diethylether. After removing the solvent, thesolid was dried at 110° C. overnight. The octyl-bonded silica contained10.9% (by weight) of carbon, which corresponds to a surface coverage ofabout 3.7 μmole of octyl groups per square meter. The use of anequivalent amount of the olefin-platinum complex dicyclopentadienylplatinum(II) dichloride as catalyst resulted in essentially the samelevel of surface coverage. A partial IR spectrum of the product is shownin curve A of FIG. 2.

A similar procedure, this time with 1-octadecene (density 0.79 g/cc, 99%purity) instead of 1-octene, was followed to prepare an octadecyl-bondedsilica. A carbon content of 11.8% (by weight) was obtained whichcorresponded to a surface coverage of about 1.8 μm/m². Curve B of FIG. 2shows a partial IR spectrum of the octadecyl-silica product.

EXAMPLE 4 Long-Term Hydrolysis Test of an Octyl-Silica PreparedAccording to the Invention

Using Vydac 101TPB™ silica as a substrate, an octyl-bonded silica wasprepared by consecutively applying the procedures described in examples1 and 3.

For the hydrolysis test, 0.75 g of the bonded phase material weresuspended in 1 mL of dioxane by magnetically stirring for 5 minutes.Then, 40 mL of an aqueous 15 mM trifluoroacetic acid or 15mM phosphatepH 2.0 solution containing 10% v/v dioxane were carefully added. Themixture was magnetically agitated at room temperature for 12 hours.After this period, a 2-mL aliquot of the well-agitated suspension wastaken and the liquid of the remaining mother suspension was removed bycentrifugation. A fresh treating solution was added and the hydrolysiscontinued for a new 12-hour period. After each sampling, the volume ofthe treating solution is decreased so as to maintain a constantliquid-to-solid ratio during the entire process. The procedure isrepeated over a total time of about 100 hours. The silica from each 2-mLaliquot sample was washed consecutively with 3-mL portions of 1:1 v/vTHF/water, THF and finally diethylether. The solid was dried at 110° C.under vacuum for several hours and its remaining carbon contentdetermined by a conventional combustion method. The decrease in carboncontent (% by weight), or its corresponding molar surface coverage(μmoles/m²), is a direct measure of the loss of bonded material from thesupport.

For comparison purposes, a parallel test was also instituted on acommercially prepared (via a silanization procedure according to thecurrent art) octyldimethylsilyl-silica. The starting silica support wasthe same for both the commercial batch and the product of thisinvention. The plots in FIGS. 3-6 clearly show that the rate ofdegradation of the silica modified via hydrosilation (present invention)is significantly lower than that of the same substrate modified viasilanization (currently available art). At the end of the test, thecommercial product had lost about 50% of its initial coverage while,under identical conditions, the hydrosilation product lost only about15% of its starting coverage material. The improved hydrolytic stabilityof the product from the present invention over the product from thecurrent art is believed due to the superior strength of the Si--Clinkage, as compared to that of prior art Si--Si--C linkages.

It is to be understood that while the invention has been described abovein conjunction with preferred specific embodiments, the description andexamples are intended to illustrate and not to limit the scope of theinvention which is defined by the scope of the appended claims.

We claim:
 1. A solid intermediate, useful in chromatographic orelectrophoretic separations after derivatization, comprising:aninorganic oxide substrate defining a surface and having silicon hydridegroups attached to the surface, the surface being exposable to fluidswith components therein being separated.
 2. The solid intermediate as inclaim 1 wherein the inorganic oxide substrate is selected from the groupconsisting of oxides of silicon, aluminum, zirconium, tin, titanium, andcombinations thereof.
 3. The solid intermediate as in claim 1 whereinthe substrate is a silicon oxide.
 4. The solid intermediate as in claim1 wherein the substrate is particulate silica or fused silica.
 5. Thesolid intermediate as in claim 4 wherein the particulate silica isporous.
 6. The solid intermediate as in claim 4 wherein the fused silicais formed as a capillary tube.
 7. The solid intermediate as in claim 6wherein the capillary tube is adapted for gas chromatography or forcapillary zone electrophoresis.
 8. A solid intermediate useful inchromatographic or electrophoretic separations after derivatization,prepared by the process comprising:providing a solid inorganic oxidesubstrate defining a surface and having surface hydroxyl groups and,converting the surface hydroxyl groups to intermediate surface siliconhydride groups by either (a) when the surface hydroxyl groups aresilanol groups sequentially halogenating the surface silanol groups andreducing the halogenated moieties to form the silicon hydride groups, or(b) reacting the surface hydroxyl groups with a trihydroxyhydridosilanehydrolysis product from a trisubstituted silane.
 9. A solid support,useful in chromatographic or electrophoretic separations, prepared bythe process comprising:providing a solid intermediate defining a surfaceand having surface hydroxyl groups; converting the surface hydroxylgroups to intermediate surface silicon hydride groups by either (a) whenthe surface hydroxyl groups are silanol groups sequentially halogenatingthe surface silanol groups and reducing the halogenated moieties to formthe silicon hydride groups, or (b) reacting the surface hydroxyl groupswith a trihydroxyhydridosilane hydrolysis product from a trisubstitutedsilane; and reacting the silicon hydride groups of said intermediatewith a reagent containing at least one terminal unsaturated hydrocarbongroup, in the presence of a metal catalyst.
 10. The support as in claim9 wherein the terminal unsaturated hydrocarbon group is --CH═CH₂ or--C.tbd.CH.
 11. The support as in claim 9 wherein the reagent furthercomprise reactive functionalities selected from the group consisting ofhydrocarbons, substituted hydrocarbons, carbonyls, carboxyls, esters,amines, amides, sulfonic acids, and epoxides.
 12. A method for preparinga solid support useful for chromatographic and electrophoreticseparations comprising:providing a solid inorganic substrate defining asurface and having surface hydroxyl groups; and converting the surfacehydroxyl groups of said substrate to intermediate surface siliconhydride groups by either (a) when the surface hydroxyl groups aresilanol groups sequentially halogenating the surface silanol groups andreducing the halogenated moieties to form the silicon hydride groups, or(b) reacting the surface hydroxyl groups with a trihydroxyhydridosilanehydrolysis product from a trisubstituted silane.
 13. The method as inclaim 12 further comprising:reacting the intermediate "surface siliconhydride groups" with a reagent containing at least one terminalunsaturated hydrocarbon group, in the presence of a metal catalyst. 14.The method as in claim 12 wherein the terminal unsaturated hydrocarbongroup is --CH═CH₂ or --C.tbd.CH.
 15. The method as in claim 12 whereinthe reagent further comprises reactive functionalities selected from thegroup consisting of hydrocarbons, substituted hydrocarbons, carbonyls,carboxyls, esters, amines, amides, sulfonic acids, and epoxides.